Paeoniflorin Promotes Ovarian Development in Mice by Activating Mitophagy and Preventing Oxidative Stress

During the development of animal organs, various adverse stimuli or toxic environments can induce oxidative stress and delay ovarian development. Paeoniflorin (PF), the main active ingredient of the traditional Chinese herb Paeonia lactiflora Pall., has protective effects on various diseases by preventing oxidative stress. However, the mechanism by which PF attenuates oxidative damage in mouse ovaries remains unclear. We evaluated the protective effects of PF on ovaries in an H2O2-induced mouse oxidative stress model. The H2O2-induced mouse ovarian oxidative stress model was used to explore the protective effect of PF on ovarian development. Histology and follicular development were observed. We then detected related indicators of cell apoptosis, oxidative stress, and autophagy in mouse ovaries. We found that PF inhibited H2O2-induced ovarian cell apoptosis and ferroptosis and promoted granulosa cell proliferation. PF prevented oxidative stress by increasing nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression levels. In addition, the autophagic flux of ovarian cells was activated and was accompanied by increased lysosomal biogenesis. Moreover, PF-mediated autophagy was involved in clearing mitochondria damaged by H2O2. Importantly, PF administration significantly increased the number of primordial follicles, primary follicles, secondary follicles, and antral follicles. PF administration improved ovarian sizes compared with the H2O2 group. The present study suggested that PF administration reversed H2O2-induced ovarian developmental delay and promoted follicle development. PF-activated mitophagy is crucial for preventing oxidative stress and improving mitochondrial quality.


Introduction
The ovaries play a key role in maintaining female reproductive capacity.Exposure to toxic environments or stress stimuli can cause ovarian oxidative stress.Oxidative stress is a state of intracellular oxidation and antioxidant imbalance and is considered to be an important factor in damaging reproductive performance [1,2].The main source of reactive oxygen species (ROS) in cells is mitochondria [3].Mitochondrial dysfunction is generally considered to be responsible for the excessive accumulation of ROS.The excessive accumulation of ROS further damages the mitochondria and ultimately accelerates the apoptosis process.Oxidative stress causes the excessive production of ROS and damages cell mitochondrial function.An increasing number of studies have shown that the ovaries are susceptible to oxidative stress during development [4][5][6].Many studies have confirmed that oxidative stress contributes to female infertility [1,7,8].Oxidative stress in the ovary can delay follicular development, cause follicular atresia, and lead to female infertility [8].Shen et al. [9] suggested that oxidative stress impairs granulosa cell function in mice and induces apoptosis.However, the detailed mechanisms by which oxidative stress induces arrested ovarian development and follicular atresia remain largely unknown.
Autophagy is an important mechanism for cells to maintain homeostasis and can participate in the removal of damaged organelles and proteins.To maintain mitochondrial quality, cells are able to selectively remove damaged mitochondria by mitophagy.Normal mitochondrial activity is crucial to cell function, and the timely elimination of damaged mitochondria is a self-protection mechanism in cells.Mitophagy prevents damaged mitochondria from further damaging cells.PTEN-induced kinase 1 (PINK1) interacts with Parkin to jointly regulate the mitophagy process to maintain mitochondrial quality.Jiang et al. found that melatonin inhibits the PINK1-Parkin pathway and mitophagy to protect mouse granulosa cells from oxidative damage [10].Adverse stimuli such as starvation, organelle damage, and oxidative stress can induce an increase in autophagy activity.Oxidative stress can cause excessive production of ROS and induce autophagy in granulosa cells [11].Furthermore, Zhang et al. [12] reported that oxidative stress induces follicular atresia and reduces oocyte developmental capacity in mice.Autophagy of germ cells in mouse ovaries is critical to maintaining the primordial follicle pool [13,14].Gioia et al. [15] suggested that autophagy inhibits apoptosis in porcine granulosa cells.These results indicate that autophagy plays a dual role in oxidative damage and ovarian development.However, the regulatory role of autophagy in H 2 O 2 -induced ovarian oxidative stress still needs further study.
Paeoniflorin (PF) is the principal bioactive constituent isolated from the traditional Chinese herb (Paeonia lactiflora Pall.).PF has anti-inflammatory, immunomodulatory, and antioxidative effects [16][17][18].Research has demonstrated that PF inhibits oxidative stress and autophagy through the Nrf2/HO-1 signaling pathway in human umbilical vein endothelial cells [19].In addition, Wang et al. found that PF activates the Akt/Nrf2/GPX4 pathway to prevent ferroptosis [20].In the rat polycystic ovarian syndrome model, PF treatment decreased TGF-β1 and Smad3 expression levels and delayed ovarian fibrosis [21].Wu et al. [22] reported that PF administration improves the ovarian index and follicle development in diminished ovarian reserve mice and promotes the synthesis of estradiol in ovarian granulosa-like KGN cells.Paeonia lactiflora Pall., a traditional Chinese medicine, is mostly used to improve female fertility and ovarian function [23].Paeoniflorin is the main active ingredient of Paeonia lactiflora Pall., but its regulatory role and molecular mechanism in ovarian oxidative stress have not been determined.
In this study, the H 2 O 2 -induced mouse ovarian oxidative stress model was used to explore the protective effect of PF on ovarian development under H 2 O 2 -induced oxidative damage and to clarify its underlying mechanism.We found that PF effectively improved the development of ovaries and follicles in H 2 O 2 -induced oxidative damage in mouse ovaries by activating autophagy, inhibiting oxidative stress, and reducing cell apoptosis.

H 2 O 2 -Induced Ovarian Developmental Delay in Mice Is Attenuated by PF Administration
To determine the protective effect of PF on ovarian development, we established an oxidative stress model targeting mouse ovaries using H 2 O 2 .After H 2 O 2 and PF treatment of 3-week-old mice for 7 weeks, ovarian tissues from each group were collected (Figure 1A).The results showed that the body weight of mice did not change significantly in the H 2 O 2 and PF + H 2 O 2 groups (p > 0.05) compared with the control group (Figure 1B).After H 2 O 2 treatment, the ovary weight and ovary weight/body weight significantly decreased (p < 0.05), while PF administration reversed the damage due to H 2 O 2 treatment (p < 0.05, Figure 1C,D).Further results revealed that H 2 O 2 -treated mice displayed more impaired ovarian follicle structures than mice in the control and PF + H 2 O 2 groups.PF treatment promoted follicle development and improved ovarian sizes compared with the H 2 O 2 group (Figure 1E).Moreover, we counted the number of primordial follicles, primary follicles, secondary follicles, antral follicles, and atretic follicles in histological sections of ovaries for each group.H 2 O 2 -induced oxidative damage resulted in a significant reduction (p < 0.05) in the number of primordial follicles, primary follicles, secondary follicles, and antral follicles (Figure 1F-I).In contrast, PF administration significantly increased (p < 0.05) the number of follicles of various types and improved follicle development (Figure 1F-I).However, no significant changes were found (p > 0.05) in the number of atretic follicles in each treatment group (Figure 1J).The results suggested that PF administration reversed H 2 O 2 -induced ovarian developmental delay in mice.
promoted follicle development and improved ovarian sizes compared with the H2O2 group (Figure 1E).Moreover, we counted the number of primordial follicles, primary follicles, secondary follicles, antral follicles, and atretic follicles in histological sections of ovaries for each group.H2O2-induced oxidative damage resulted in a significant reduction (p < 0.05) in the number of primordial follicles, primary follicles, secondary follicles, and antral follicles (Figure 1F-I).In contrast, PF administration significantly increased (p < 0.05) the number of follicles of various types and improved follicle development (Figure 1F-I).However, no significant changes were found (p > 0.05) in the number of atretic follicles in each treatment group (Figure 1J).The results suggested that PF administration reversed H2O2-induced ovarian developmental delay in mice.

PF Inhibits H2O2-Induced Cell Apoptosis and Promotes Granulosa Cell Proliferation
An increase in the number of apoptotic cells can lead to a reduction in organ size.To explore whether the reduction in ovarian size caused by H2O2 is related to ovarian cell apoptosis, we analyzed ovarian cell proliferation and apoptosis.IF staining results showed that the PCNA-positive signal was specifically localized in granulosa cells (Figure 2A).H2O2 treatment significantly decreased (p < 0.05) the PCNA fluorescence intensity in ovaries, while PF administration significantly increased (p < 0.01) the expression level of PCNA (Figure 2B).Moreover, Western blot results showed that PF administration decreased the expression level of BAX (p < 0.05) and increased the BCL2 expression level (p < 0.01) in the ovaries (Figure 2C-E).PF administration consistently, significantly reduced

PF Inhibits H 2 O 2 -Induced Cell Apoptosis and Promotes Granulosa Cell Proliferation
An increase in the number of apoptotic cells can lead to a reduction in organ size.To explore whether the reduction in ovarian size caused by H 2 O 2 is related to ovarian cell apoptosis, we analyzed ovarian cell proliferation and apoptosis.IF staining results showed that the PCNA-positive signal was specifically localized in granulosa cells (Figure 2A).H 2 O 2 treatment significantly decreased (p < 0.05) the PCNA fluorescence intensity in ovaries, while PF administration significantly increased (p < 0.01) the expression level of PCNA (Figure 2B).Moreover, Western blot results showed that PF administration decreased the expression level of BAX (p < 0.05) and increased the BCL2 expression level (p < 0.01) in the ovaries (Figure 2C-E

PF Prevents Oxidative Stress and Inhibits Ferroptosis in Mouse Ovaries
To determine whether PF prevented H2O2-induced oxidative stress to promote cell survival, we analyzed the expression of Nrf2 and HO-1 in mouse ovaries.IF staining showed that in the PF + H2O2 group, PF administration significantly increased (p < 0.05) Nrf2 and HO-1 expression levels compared with the H2O2 group (Figure 3A-C).The results of Western blot showed that H2O2 treatment decreased the GPX4 expression level (p < 0.01) compared with the control group, and PF administration caused a significant increase (p < 0.01) in the GPX4 expression level in the PF + H2O2 group (Figure 3D,E), indicating that PF improved the ability of ovarian cells to resist ferroptosis.The results suggested that H2O2-induced oxidative stress and ferroptosis in ovarian cells were inhibited by PF.

PF Prevents Oxidative Stress and Inhibits Ferroptosis in Mouse Ovaries
To determine whether PF prevented H 2 O 2 -induced oxidative stress to promote cell survival, we analyzed the expression of Nrf2 and HO-1 in mouse ovaries.IF staining showed that in the PF + H 2 O 2 group, PF administration significantly increased (p < 0.05) Nrf2 and HO-1 expression levels compared with the H 2 O 2 group (Figure 3A-C).The results of Western blot showed that H 2 O 2 treatment decreased the GPX4 expression level (p < 0.01) compared with the control group, and PF administration caused a significant increase (p < 0.01) in the GPX4 expression level in the PF + H 2 O 2 group (Figure 3D,E), indicating that PF improved the ability of ovarian cells to resist ferroptosis.The results suggested that H 2 O 2 -induced oxidative stress and ferroptosis in ovarian cells were inhibited by PF.

PF Activates Autophagy Activity and Lysosomal Biogenesis
Autophagy plays a very important role in oxidative stress.To explore whether autophagy is involved in the protective effect of PF against ovarian oxidative damage, we detected the expression levels of autophagy-related proteins.The results showed that H2O2 significantly decreased (p < 0.05) the LC3-II expression level and promoted p62 protein accumulation (p < 0.05, Figure 4A-C), indicating that H2O2-induced oxidative stress reduced autophagy activity in ovarian cells.PF treatment significantly upregulated LC3-II expression level (p < 0.01), leading to a decrease in the p62 protein level (Figure 4A-C).IF staining consistently showed that PF treatment increased the Beclin-1 fluorescence intensity (p < 0.01) and decreased p62 fluorescence intensity (p < 0.05) compared with the H2O2 group (Figure 4D-F).Furthermore, the TFEB expression level was significantly upregulated (p < 0.01) in ovaries after PF treatment (Figure 4G,H).PF administration increased (p < 0.05) LAMP2 expression level in the PF+ H2O2 group (Figure 4I,J), suggesting that PF administration activated autophagy activity and lysosomal biogenesis in ovarian cells.

PF Activates Autophagy Activity and Lysosomal Biogenesis
Autophagy plays a very important role in oxidative stress.To explore whether autophagy is involved in the protective effect of PF against ovarian oxidative damage, we detected the expression levels of autophagy-related proteins.The results showed that H 2 O 2 significantly decreased (p < 0.05) the LC3-II expression level and promoted p62 protein accumulation (p < 0.05, Figure 4A-C), indicating that H 2 O 2 -induced oxidative stress reduced autophagy activity in ovarian cells.PF treatment significantly upregulated LC3-II expression level (p < 0.01), leading to a decrease in the p62 protein level (Figure 4A-C).IF staining consistently showed that PF treatment increased the Beclin-1 fluorescence intensity (p < 0.01) and decreased p62 fluorescence intensity (p < 0.05) compared with the H 2 O 2 group (Figure 4D-F).Furthermore, the TFEB expression level was significantly upregulated (p < 0.01) in ovaries after PF treatment (Figure 4G,H).PF administration increased (p < 0.05) LAMP2 expression level in the PF+ H 2 O 2 group (Figure 4I,J), suggesting that PF administration activated autophagy activity and lysosomal biogenesis in ovarian cells.

PF Promotes Mitophagy to Improve Mitochondrial Quality
Oxidative stress can cause mitochondrial dysfunction, leading to cell apoptosis.To determine whether mitochondrial function is affected by H 2 O 2 and PF, mitochondriarelated protein expression levels were detected.Western blot results showed that H 2 O 2 treatment significantly increased (p < 0.05) the expression levels of fusion protein MFN1, fission protein DRP1, and mitochondrial membrane protein TOM20.At the same time, PF administration significantly decreased (p < 0.05) the expression levels of these proteins (Figure 5A-D

PF Promotes Mitophagy to Improve Mitochondrial Quality
Oxidative stress can cause mitochondrial dysfunction, leading to cell apoptosis.To determine whether mitochondrial function is affected by H2O2 and PF, mitochondria-related protein expression levels were detected.Western blot results showed that H2O2 treatment significantly increased (p < 0.05) the expression levels of fusion protein MFN1, fission protein DRP1, and mitochondrial membrane protein TOM20.At the same time, PF admin-

Discussion
Oxidative stress can delay follicular development and reduce ovarian function, leading to reduced female reproductive capacity and even infertility.More and more adverse factors or toxic substances cause oxidative stress and affect ovarian function.PF, as the main active ingredient of the traditional Chinese herb Paeonia lactiflora Pall., can improve ovarian function.In this study, we found that (1) PF protected ovarian cells from H2O2induced oxidative damage and inhibited ferroptosis; (2) PF reduced the amount of ovarian cell apoptosis and promoted ovarian development; (3) PF activated mitophagy to maintain mitochondrial quality control in ovaries.
Oxidative stress is a phenomenon that is an imbalance between the production of ROS and the antioxidant defenses of cells.Many studies have demonstrated that female infertility is related to oxidative stress [1,24,25].The H2O2-induced oxidative stress model is widely used to explore the molecular mechanisms related to oxidative damage.The present study found that H2O2 caused oxidative stress, while PF treatment increased the expression levels of Nrf2 and HO-1, which is consistent with previous study [16].Nrf2 is a crucial regulator of cellular antioxidant capacity, but its overactivation also favors cancer progression.Previous studies have suggested that increasing the Nrf2 expression level can improve ovarian antioxidant capacity and promote ovarian development [26,27].Ren

Discussion
Oxidative stress can delay follicular development and reduce ovarian function, leading to reduced female reproductive capacity and even infertility.More and more adverse factors or toxic substances cause oxidative stress and affect ovarian function.PF, as the main active ingredient of the traditional Chinese herb Paeonia lactiflora Pall., can improve ovarian function.In this study, we found that (1) PF protected ovarian cells from H 2 O 2 -induced oxidative damage and inhibited ferroptosis; (2) PF reduced the amount of ovarian cell apoptosis and promoted ovarian development; (3) PF activated mitophagy to maintain mitochondrial quality control in ovaries.
Oxidative stress is a phenomenon that is an imbalance between the production of ROS and the antioxidant defenses of cells.Many studies have demonstrated that female infertility is related to oxidative stress [1,24,25].The H 2 O 2 -induced oxidative stress model is widely used to explore the molecular mechanisms related to oxidative damage.The present study found that H 2 O 2 caused oxidative stress, while PF treatment increased the expression levels of Nrf2 and HO-1, which is consistent with previous study [16].Nrf2 is a crucial regulator of cellular antioxidant capacity, but its overactivation also favors cancer progression.Previous studies have suggested that increasing the Nrf2 expression level can improve ovarian antioxidant capacity and promote ovarian development [26,27].
Ren et al. [16] reported that PF activates the Nrf2/HO-1 signaling pathway to improve antioxidant capacity in H9c2 cells.The pterostilbene-activated Nrf2/HO-1 signaling pathway protected human ovarian granulosa cells from oxidative stress and ferroptosis [28].Spermidine consistently inhibits oxidative stress and ferroptosis and alleviates ovarian damage by the Nrf2/HO-1/GPX4 pathway [26].These studies suggest that Nrf2 plays a critical role in ovarian resistance to oxidative damage.In addition, GPX4 is a key enzyme that clears lipid peroxides, and its expression level is regulated by the transcription factor Nrf2 [29].Ma et al. [30] confirmed that PF inhibits ferroptosis in a mouse acute kidney injury model and human renal tubular epithelial cells.We observed that PF elevated the GPX4 protein level in ovarian tissues, consistent with the results of a previous study [30], which indicated that PF protected the ovary against oxidative stress by activating the Nrf2/HO-1 signaling pathway.
In this study, H 2 O 2 -induced oxidative stress delayed ovarian development and reduced ovarian size.Previous study has suggested that reduced organ size is associated with increased numbers of apoptotic cells [31].Apoptosis plays an important role in ovarian function and follicle development [32].BAX is a pro-apoptotic protein, while BCL2 is an anti-apoptotic protein.We found that H 2 O 2 treatment upregulated BAX expression levels and downregulated BCL2 expression levels, which indicated that H 2 O 2 caused excessive apoptosis of ovarian cells.Therefore, oxidative stress caused by H 2 O 2 delays ovarian development possibly by increasing ovarian cell apoptosis, and apoptosis caused by H 2 O 2 is the direct cause of the reduction in ovarian size.Under oxidative stress conditions, PF prevented oxidative damage and reduced the number of ovarian apoptotic cells.Notably, H 2 O 2 -reduced ovarian size was reversed by PF.Our results are consistent with previous studies [33,34], which suggested that PF treatment can inhibit ovarian cell apoptosis and promote ovarian development.Furthermore, we found that PF increased PCNA expression levels in the ovary.PCNA is an important indicator for detecting cell proliferation status.Interestingly, PCNA was specifically localized in granulosa cells within follicles in this study, which may be related to the massive proliferation of granulosa cells during follicle development [35,36].A previous study demonstrated that PF inhibits proliferation in breast cancer cells [37].Wang et al. [38] also found that PF inhibits proliferation and promotes apoptosis in colon cancer cells, which is attributed to the anti-inflammatory effect of PF.However, we observed that PF promoted granulosa cell proliferation and follicle development in H 2 O 2 -induced ovarian oxidative damage.The results of this study are different from those of previous studies, which may be due to the previous studies focusing on cancer cells or mouse models [37,38].These findings indicate that the protective effects and mechanisms of PF vary in different cells or organs.Previous studies generally believe that apoptosis is the main factor of follicular atresia [39,40].Further results showed that there was no significant difference in the amount of follicle atresia before and after PF treatment (p > 0.05), indicating that PF can accelerate follicle development and inhibit ovarian cell apoptosis in the ovary, but it does not participate in regulating the process of follicular atresia.
Oxidative stress damages mitochondria, causing mitochondrial dysfunction.Cao et al. [41] confirmed that PF reduces ROS levels and increases mitochondrial membrane potential to restore the mitochondrial quality in macrophages.In this study, we observed consistent results with Cao et al. [41] that H 2 O 2 -induced oxidative stress caused mitochondrial dysfunction, while PF repaired H 2 O 2 -damaged mitochondria.Delayed ovarian development is accompanied by impaired autophagy.It is well known that autophagy plays a critical role in clearing damaged mitochondria.In recent years, many studies have confirmed that PF is involved in the regulation of autophagy activity [41][42][43].LC3-II is used to analyze autophagy activity and is localized on the autophagosome membrane [44].Beclin-1 participates in the extension of autophagosomes.p62, an autophagy protein substrate, was used to monitor autophagic flux [45].The present study showed that PF upregulated LC3-II and Beclin-1 expression levels and reduced p62 protein accumulation, activating autophagic flux in ovarian cells.And PF increased TFEB and LAMP2 expression levels, suggesting that lysosomal biogenesis was increased, and autophagic turnover was normal.Our findings are consistent with those of Zhou et al. [46] that PF enhances autophagy activity to prevent adverse stimuli and promote cell survival.Sun et al. [47] reported that PF increases LC3 expression, enhances Parkin-mediated mitophagy, and rescues mitochondrial damage in PC12 cells.Our study found that PF upregulated mitophagy marker PINK1 and Parkin expression levels and increased the co-localization of LC3 and COX IV, which is consistent with Sun et al. [47].A large number of studies have demonstrated that autophagy can prevent oxidative stress and improve cell function [48][49][50].These findings indicate that PF-activated mitophagy clears damaged mitochondria and improves mitochondrial quality, thereby improving ovarian cell function.In the H 2 O 2induced ovarian oxidative stress model, oxidative damage is accompanied by impaired mitophagy, which further aggravates ovarian dysfunction, whereas PF activates mitophagy to resist oxidative stress and thereby promotes ovarian development.

Ethics Statement
All procedures involving animals were approved and conducted by the Institutional Animal Care and Use Committee of Zhejiang A&F University, China (ZAFUAC2023005).

Animals
Thirty female ICR mice (3 weeks old) were kept at 22 ± 2 group.The same volume of normal saline was injected into the control group.After 7 weeks of treatment, the mice were weighed and euthanized.Ovarian tissues were collected and immediately weighed.The tissues were divided into two parts: (1) the tissues of part one were fixed in Bouin's solution for histological assays; (2) the tissues of part two were frozen for Western blot.

Histology and Follicle Count
The fixed ovarian tissues were embedded and sliced (5 µm).Hematoxylin and eosin staining or histological observation was performed as described previously [51].The images were captured with a microscope (CKX53SF, OLYMPUS, Tokyo, Japan).Serial sections of the ovaries were performed to count the number of follicles.The number of follicles at each stage was observed and counted under a microscope.Primordial follicles: a layer of flattened granulosa cells surrounding the oocyte.Primary follicles: the flat granulosa cells surrounding the oocyte become columnar granulosa cells.Secondary follicles: one layer of granulosa cells surrounding the oocyte becomes two to three layers; no follicular cavity.Antral follicles: multiple layers of granulosa cells surrounding the oocyte; clear follicular cavity.

TUNEL Assay
The apoptosis of ovarian cells was evaluated by TUNEL staining according to the manufacturer's instructions.4',6-diamidino-2-phenylindole (DAPI, 1 µg/mL) was used to label the nuclear factor.The images were captured with a fluorescence microscope (CKX53SF, OLYMPUS, Tokyo, Japan).Six sections were randomly selected to detect cell apoptosis.

Statistical Analysis
The data are presented as mean ± standard error of the mean (SEM).Statistical tests were performed using GraphPad Prism software (version 6).Differences were determined using a one-way analysis of variance (ANOVA).p < 0.05 was considered statistically significant.The experiments were repeated three times.

Conclusions
In summary, we confirmed that PF plays a protective effect in the ovarian development of the H 2 O 2 -induced mouse oxidative stress model.PF prevented oxidative stress, promoted granulosa cell proliferation, and thereby promoted follicle development.Furthermore, PF improved mitochondrial quality by activating mitophagy activity.Thus, our study provides new evidence that PF promotes ovarian development, and provides a theoretical basis for improving female reproductive capacity.
). PF administration consistently, significantly reduced (p < 0.01) the number of TUNEL-positive granulosa cells in the PF + H 2 O 2 group compared with the H 2 O 2 group (Figure 2F,G).PF administration significantly decreased (p < 0.05) the γH2A fluorescence intensity in the PF + H 2 O 2 group (Figure 2H,I), indicating that H 2 O 2 -induced DNA damage was rescued by PF administration.The results suggested that PF treatment inhibited cell apoptosis and promoted granulosa cell proliferation.
(p < 0.01) the number of TUNEL-positive granulosa cells in the PF + H2O2 group compared with the H2O2 group (Figure2F,G).PF administration significantly decreased (p < 0.05) the γH2A fluorescence intensity in the PF + H2O2 group (Figure2H,I), indicating that H2O2induced DNA damage was rescued by PF administration.The results suggested that PF treatment inhibited cell apoptosis and promoted granulosa cell proliferation.
). Mitophagy can clear damaged mitochondria to maintain mitochondrial quality.We further analyzed mitophagy levels in different treatment groups.IF staining showed that PF administration increased the fluorescence intensities of PINK1 and Parkin in the PF + H 2 O 2 group compared with the H 2 O 2 group (p < 0.05, Figure5E-G).Importantly, PF administration markedly increased the co-localization of LC3 and COX IV (Figure5H-J), indicating that PF promoted mitophagy to maintain mitochondrial quality in oxidative damage to the ovary induced by H 2 O 2 .

Table 1 .
Antibodies used in this study.